1. Field of the Invention
The present invention relates to a method for screening for an agonist or antagonist participating in vasodilating action, and compounds obtained by the method. More specifically, the invention relates to a method for screening for an agonist or antagonist using a receptor of maxadilan, and a compound which specifically binds to the receptor, and further, according to circumstances, promotes the cAMP production of a culture cell having a certain cAMP production ability.
2. Description of the Related
Maxadilan is known to exist i n the sialaden of the sand fly Lutzomyia longipalpis which is a sucking insect. When applied on the epidermis of animals, maxadilan causes erythema without itch and pain, and is considered to be a strong vasodilator. This biological action is known to be very analogous to the action of calcitonin gene-related peptide (CGRP).
Purification of maxadilan from the salivary glands of the sand fly; the nucleotide sequence encoding its amino acid sequence; and cloning of the gene comprising the nucleotide sequence; and expression of the recombinant maxadilan are announced, for example, in WO 91/00293 and E. A. Lerner et al., J. Bio. Chem. 267, 1062-1066 (1992). Lerner et al. cloned a DNA encoding a fused protein of the recombinant maxadilan with glutathione S-transferase (GST); expressed the fused protein; and cleaved the fused protein with factor Xa; and thereby obtained the recombinant maxadilan as a product in which a peptide fragment (GILxe2x88x92) consisting of three amino acid residues binds to the N-terminal cysteine of maxadilan (the first cysteine of natural maxadilan; namely corresponding to Cys (1) of SEQ ID NO: 1 of the sequence listing).
Further, the present inventors have already found that a recombinant (see SEQ ID NO: 2) in which a peptide fragment (GSILxe2x88x92) binds to the N-terminal cysteine of natural maxadilan has an erythema formation activity superior to that of natural maxadilan. In the present specification, maxadilan merely referred to means the recombinant represented by SEQ ID NO: 2.
These recombinant maxadilans are common to CGRP in the point of showing a strong erythema formation activity (hereafter merely referred to as xe2x80x9cerythema activityxe2x80x9d), but draws attention in the point that they show an erythema activity of almost 500-times that of CGRP per one molecule, and the effect lasts for a longer period of time. Therefore, interest in provision of further variants of maxadilan and evaluation results of these physiological activities is never-ending.
An in vivo test using as erythema formation activity induced by intracutaneously injecting a test polypeptide into white rabbits has been chiefly used in evaluation for the activity. However, a system which is simple and capable of evaluating activities other than erythema formation activity as well as erythema formation activity, is desired. If such an evaluation system is available, it is believed that it will be easy to conduct evaluation on not only variants of maxadilan but various non-peptide compounds.
Thus, the object of the invention lies in providing an in vitro test method, said method being a method for screening for compounds capable of evaluating activities having correlation with the erythema formation activity and other activities. Further, it is also an object of the invention to provide novel compounds selected by such screening method.
The present inventors have created various variant maxadilans, and have investigated correlation between these structures and actions. Further, we found that maxadilan specifically binds to specific tissue preparations from mammals, and in certain culture cells, the production amount of cyclic adenosine 3xe2x80x2,5xe2x80x2-mono-phosphoric acid (cAMP) is enhanced by maxadilan. Among the above various variant maxadilans, there are those which have the ability to inhibit the binding of maxadilan to the tissue preparation (competitive binding ability), and moreover, enhance cAMP production amount. Thus, it was confirmed that there is a certain correlation between the binding inhibition ability and the cAMP production ability, and the erythema formation ability.
According to the invention, based on such findings, these are provided, for example, as to vasodilating action, according to circumstances, a method for screening for compounds inhibiting such action, and compounds selected by such method.
Thus, the first embodiment of the invention is a method for screening for an agonist or antagonist participating in vasodilating action, said method comprising
(A) a step of preparing a tissue preparation derived from a mammal, to which maxadilan specifically binds,
(B) a step of preparing an analyte
(C) a step of contacting the tissue preparation of the step (A) with the analyte of the step (B),
(D) a step of assaying the binding affinity of the analyte to the tissue preparation, in the step (C),
(E) when the binding affinity in the step (D) is positive, a further step of assaying the influence of the analyte having such action on the cAMP production ability of an animal culture cell to which maxadilan specifically binds and which has a cAMP production ability, and
(F) a step of determining whether the analyte is an agonist or antagonist, based on the results of the step (D) and the step (E)
The second embodiment of the invention is a mammalian preparation having sites (receptors) to which maxadilan specifically binds, the preparation being utilizable for the screening method.
The third embodiment of the invention is a variant maxadilan which specifically binds to a tissue preparation derived from a mammal, having sites to which maxadilan specifically binds, or, according to circumstances, a variant maxadilan which has the above property, and, moreover, promotes the production of cAMP in an animal culture cell which maxadilan specifically binds to and has cAMP production ability.
The thus provided variant maxadilans exhibit binding ability to the receptor of the tissue preparation (ability to competitively inhibit maxadilan), and it is considered that, among them, such a variant maxadilan having, in addition, the cAMP production promotion ability is one acting as an agonist on maxadilan receptors, and such a variant maxadilan exhibiting no cAMP production promotion ability is one acting as an antagonist on maxadilan receptors.